Plant proanthocyanidin extract effective at inhibiting adherence of bacteria with P-type fimbriae to surfaces

ABSTRACT

The present invention is directed to isolation and identification of plant proanthocyanidin extracts and particular proanthocyanidin compounds for prevention and treatment of urinary tract infections caused by P-type  Escherichia coli . These extracts can be obtained from any proanthocyanidin-containing plants, including plants of the families Ericaceae, Rosaceae, Pinaceae, Vitaceae and the like. Preferably the extracts are from cranberry plants (especially,  Vaccinium macrocarpon ) and other plants, particularly fruit and berry plants from the Vaccinium spp. The extracts and compounds are also provided as pharmaceutical compositions, food additives and food compositions, especially beverages, ground meat preparations and cranberry-containing food products. The invention also relates to methods of reducing pathogenicity of P-type  E. coli  in the digestive tracts of cattle and reducing P-type  E. coli  contamination in ground meat as well as methods of detecting P-type bacteria.

This application is a continuation-in-part of provisional applicationU.S. Ser. No. 60/058,307, filed Sep. 9, 1997.

FIELD OF THE INVENTION

The present invention is directed to isolation and identification ofplant proanthocyanidin extracts. These extracts can be obtained from anyof a variety of proanthocyanidin-containing plants including members ofthe plant families Ericaceae, Rosaceae, Pinaceae and Vitaceae, andpreferably are from cranberry plants (especially Vaccinium macrocarpon),other Vaccinium spp. and grapes (Vitus spp.) These extracts are usefulfor prevention and treatment of urinary tract infections caused byP-type fimbriated Escherichia coli as well as other uses. Extractscontaining proanthocyanidins with A-type interflavanoind bonds have beenfound to have potent bioactivity for inhibiting adherence of P-type E.coli.

BACKGROUND OF THE INVENTION

Millions of women each year are diagnosed with cystitis (bladderinfections) and pyelonephritis (kidney infections). Countless numbers ofdogs and cats also suffer from chronic urinary infections and die fromrenal infections. E. coli bacteria is the most common pathogenassociated with these infections, causing over 80% of urinary tractinfections. Over 30% of women suffer recurrent infections within a 6 to12-month period and are forced to resort to extended use of antibioticsto treat these infections. Recurrent use of antibiotics can lead topathogen resistance and result in deleterious side effects and toxicityreactions. Consequently there exists a need for safe alternativemedications (e.g., non-antibiotics) that can be used to prevent or treaturinary tract infections in both animals and humans.

Cranberry juice has been shown to reduce bacteriuria associated withurinary tract infections in humans (Avorn et al., 1994, J. Am. Med. Soc.271:751-754). The effect appears to be due to the ability of certaincranberry compounds to inhibit adhesion of type 1 (implicated in bladderinfections) and P-type (implicated in kidney infections) E. colibacterial phenotypes to human bladder epithelial cells (Sobota, 1984, J.Urol. 131:1013-1016; Schmidt & Sobota, 1988, Microbios. 55:173-181;Zafriri et al., 1989, Antimicrob. Agents Chemo. 33:92-98). Zafriri etal. (1989) reported that fructose was responsible for the inhibition oftype 1 E. coli to uroepithelial cells. Zafriri et al. also reported thatcranberry juice contained a non-dialyzable substance (or substances)which inhibited binding of P-type E. coli but failed to define thechemical nature of this inhibitor. Cranberry juice has also been shownto cause immediate inhibition of agglutination as well as loss offimbriae after long-term exposure of bacteria to the juice (Ahuja etal., 1998, J. Urol. 159:559-562).

A partially-purified anti-adherence activity from cranberry has alsobeen described (U.S. Pat. Nos. 5,474,774; 5,525,341; and 5,646,178, allto Walker et al.). This activity was obtained using acidified alcohol asan extraction solvent with whole cranberry fruit followed by separationof the activity from monomer and dimer sugars by precipitation with ametal acetate or sulfate. Upon further manipulation, the reportedactivity consisted of a fraction enriched for polyphenol and flavonoidcompounds that contained as much as 10% anthocyanins. The specificity ofthis anti-adherence activity for type 1 or P-type E. coli was notdetermined.

Walker et al. (WO 96/30033; and U.S. Pat. Nos. 5,646,178 and 5,650,432)described a series of proanthocyanidin monomers, dimers, polymers,flavonoid derivatives thereof and related compounds purported to havethe ability to interfere with bacterial adherence to a surface. Thedimers and polymers of Walker were limited to compounds having B-typeinterflavanoid linkages. However, Walker failed to provide anyexperimental data correlating biological activity with aspecifically-identified compound. The Walker method involvedalkalinizing a plant material homogenate to a pH greater than 10, atreatment which causes degradation of proanthocyanidins, andprecipitating the polyphenolic compounds (together with other materials)by addition of alcohol. This precipitate contained the proposedanti-adherence activity and was further fractionated to yield thepurified active compound. Using this process with an aqueous solution ofcommercially-available Ocean Spray cranberry powder, Walker reportedobtaining a single active compound and partially characterized thecompound but failed to provide a complete (or any) chemical structurefor this compound. Walker also failed to characterize the biologicalactivity of this compound with respect to inhibition of adherence oftype 1 or P-type E. coli. In fact, the Walker assay methods could notdistinguish between these two biological activities.

Thus, prior to the present invention, there had been no identificationof the class of bioactive compounds that inhibit P-type E. coli fromadhering to surfaces such as uroepithelial cells. In accordance with theinvention, it has been discovered that extracted mixtures ofproanthocyanidins and purified proanthocyanidins are the bioactivecompounds present in cranberry and other plants that possessanti-adherence activity against P-type E. coli.

A large variety of plants are known to contain proanthocyanidins andmethods for isolating small amounts of proanthocyanidins from severaldifferent plant species have been reported. Various purification methodsfor proanthocyanidins from plant material have been described byThompson et al. (1972, J. Chem. Soc., Perkins Trans. I. 11:1387); Joneset al. (1976, Phytochem. 15:1407-1409); Wang et al. (1978, J. Food Sci.43:1402-1404); Czochanska et al. (1980, J. Chem. Soc., Perkin Trans.I:2278-2286); Foo & Porter (1981, J. Sci. Food Agric. 32:711-716);Marwan & Nagel (1986a, J. Food Sci. 51:1009-1013); Marwan & Nagel(1986b, J. Food Sci. 51:1069-1070); Morimoto et al. (1988, Chem. Pharm.Bull. 36:33-38); Devlin, U.S. Pat. No.4,309,207; and Bomser et al.(1996, Planta Med. 62:212-216).

Accordingly, the present invention identifies proanthocyanidins as thecompounds that mediate inhibition of adherence of P-type E. coli tocellular surfaces and further provides an improved method of obtainingsubstantially pure mixtures of proanthocyanidins as well as individualproanthocyanidin compounds from the plant material of cranberries andother plants known to contain proanthocyanidins. These proanthocyanidinmixtures and individual proanthocyanidin compounds are thus useful inthe prevention and/or therapeutic treatment of urinary tract infections,particularly those infections mediated by P-type E. coli or othermicroorganisms that contain structurally-related fimbriae or moleculesinvolved in microbial adherence.

There also exists a need for an inexpensive and rapid test to identifythe type of urinary tract infection that a person has contracted.Current clinical diagnosis is based on counting the number of bacteriapresent in a patient's urine without attempting to distinguish thebacterial strain as type 1 E. coli, P-type E. coli or a mixture of both.Since kidney infections (associated more with P-type E. coli) areusually more serious than bladder infections (associated more with type1 E. coli) and require different treatment regimes, having a test todistinguish between these phenotypic strain variations would bebeneficial. Thus, an inexpensive, rapid diagnostic kit to diagnoseP-type infection would allow patients to receive the proper treatment ina more timely manner and avoid the use of ineffective medications.Accordingly, the invention also addresses this problem by providingproanthocyanidin mixtures which specifically bind to P-type E. coli asreceptor analogs or prevent P-type fimbrial induction for assay kitsdesigned to aid in the clinical diagnosis of pyelonephritis-typeinfections.

SUMMARY OF THE INVENTION

The present invention is directed to proanthocyanidin extractssubstantially free of anthocyanins and flavonols. These extracts arealso shown to be free of hydrolyzable tannins, alkaloids, lipids,carbohydrates, simple sugars, protein and amino acids, alcohols andorganic acids by chemical reagent testing. These proanthocyanidinsextracts are capable of inhibiting agglutination reactions of P-type E.coli but not type 1 E. coli. In particular, those extracts containingproanthocyanidins with at least one A-type interflavanoid bond have beenfound to be more bioactive in agglutination reactions thanproanthocyanidins linked only by B-type interflavanoid bonds.

The invention also provides a method of obtaining these proanthocyanidinextracts from the plant material from any of a variety ofproanthocyanidin-containing plants. The preferred plants are in theEricaceae, Rosaceae, Pinaceae and Vitaceae families, particularly theVaccinium species of the Ericaceae family, and the Vitis species of theVitaceae family, and most preferably from cranberries (especially fromV. macrocarpon). The method is applicable for extracting theproanthocyanidins from any portion of the plant including leaves, ripefruit and unripe fruit. The method of the invention causes substantiallyless oxidative damage and/or atructural degradation to theproanthocyanidins, i.e., is less harsh, and provides improved yields ofpurified proanthocyanidins relative to known methods.

More particularly, the method of the invention is directed to preparinga proanthocyanidin extract from a plant by (a) homogenizing plantmaterial in an aqueous extraction solvent comprising at least about 10%water but no more than about 30% water, about 10% to about 70% acetone,about 5% to about 60% methanol and about 0.05% to about 1% ascorbicacid; and subjecting that extract to further purification to allowrecovery of a substantially purified proanthocyanidin extract which iscapable of inhibiting agglutination of P-type E. coli but incapable ofinhibiting agglutination of type 1 E. coli.

Hence, a preferred embodiment of the invention relates to a method ofpreparing a proanthocyanidin extract from a Vaccinium species whichcomprises:

(a) homogenizing Vaccinium plant material in an aqueous extractionsolvent comprising at least about 10% water but no more than about 30%water, about 10% to about 70% acetone, about 5% to about 60% methanoland about 0.05% to about 1% ascorbic acid to prepare a first extract;

(b) clarifying and obtaining the supernatant from the first extract;

(c) removing the solvent from the supernatant and resuspending theresidue in distilled water;

(d) subjecting the resuspended residue solution to further purificationby either

(i) applying the residue solution to reverse-phase lipophilicchromatography material equilibrated in distilled water and successivelywashing the lipophilic chromatography material with distilled water toremove sugars, with about 15% aqueous methanol to remove acids and withabout 100% acidified methanol to elute polyphenolic compounds, andremoving solvent from the polyphenolic fraction to obtain a first driedpolyphenolic fraction, or

(ii) extracting the residue solution with a non-polar extractionsolvent, recovering the aqueous phase and removing solvent therefrom toobtain a second dried fraction;

(e) suspending the first or second dried fraction in about 50% aqueousethanol, applying that solution to mixed hydrophilic-lipophilicchromatography material equilibrated in about 50% aqueous ethanol, andwashing the mixed hydrophilic-lipophilic chromatography material withabout 50% aqueous ethanol to remove non-proanthocyanidin polyphenoliccompounds; and

(f) eluting the mixed hydrophilic-lipophilic chromatography materialwith about 70% aqueous acetone to obtain the proanthocyanidin extract.This proanthocyanidin extract inhibits the agglutination of P-type E.coli but does not inhibit agglutination of type 1 E. coli.

The proanthocyanidin extract can be further fractionated using HPLC orother techniques to identify and characterize specific proanthocyanidincompounds that have anti-adherence activity against P-type E. coli orother microorganisms.

Such compounds include proanthocyanidin compounds having an average ofat least four to about seven epicatechin flavanoid units, wherein atleast two of the units are linked together by an A-type doubleinterflavanoid bond between C4 and C8 and between C2 and the oxygen ofC7 and the remaining units are linked to each other by a B-typeinterflavanoid bond between C4 and C8 or between C4 and C6.

Another aspect of the invention relates to methods of preventing ortreating urogenital infections in a mammal by administering aproanthocyanidin composition comprising the proanthocyanidin extract, aproanthocyanidin compound, a proanthocyanidin polymer or a mixturethereof, to the mammal in an amount and for a time sufficient toprevent, reduce or eliminate the symptoms associated with suchinfections and thereby lead to an amelioration or curing of theinfection. Preferably the mammal undergoing treatment is a human, butthe method is also applicable to animals, especially domesticatedanimals such as cats and dogs and livestock animals such as cattle.

As used herein a “proanthocyanidin composition” comprises aproanthocyanidin extract of the invention, a proanthocyanidin compoundof the invention, a proanthocyanidin polymer or a mixture thereof. Theproanthocyanidin composition can be provided as a pharmaceuticalcomposition, e.g., in pill form, as a food additive, e.g. for abeverage, or as a food composition. When a proanthocyanidin compositionof the invention is provided in a cranberry juice beverage, theproanthocyanidins can enhance the cranberry juice's known benefits forpreventing and treating urinary tract infections.

Hence, pharmaceutical compositions are provided which comprise aproanthocyanidin composition, including pharmaceutically-acceptablesalts of any of the proanthocyanidin compounds or polymers, with apharmaceutically acceptable carrier. In some instances, it may bepreferable to provide the therapeutic dosage in the form of a foodadditive in a beverage such as a cranberry juice-based beveragecontaining additional proanthocyanidins. The invention also providesfood compositions comprising a proanthocyanidin composition, includingpharmaceutically-acceptable salts of the compounds or polymers, mixedwith a consumable carrier. Consumable carriers include, but are notlimited to, livestock feed, domestic animal feed and consumable foodproducts, especially a cranberry-containing food products. These foodcompositions are also useful to prevent or treat urinary tractinfections.

In another embodiment, the proanthocyanidin composition of the inventioncan be used to reduce the pathogenesis of P-type E. coli found in thedigestive tracts of cattle. Such a method may be useful in decreasingcontamination of ground meat prepared from such cattle.

Yet another aspect of the invention relates to a method of detectingP-type E. coli in a body fluid, especially in urine, and use of thatmethod in diagnosis of pyelonephritis or other urinary tract infectionassociated with P-type E. coli. A kit for detecting P-type E. coli isalso provided.

A still further aspect of the invention is directed to methods ofreducing the incidence of infection after surgery, treating topicalwounds and acne, and preventing or eliminating oral infections using theproanthocyanidin composition of the invention.

Further still, the proanthocyanidin composition of the invention canalso be used as a food additive to confer protection against E. colipresent in certain food products such as ground meat and unpasteurizedjuices, or as a feed additive to reduce the pathogenesis of P-type E.coli found in the digestive tracts of animals, especially cattledestined for slaughter. When used as a food additive for ground meat, itis preferable to add the proanthocyanidin composition to the meatpreparations before or during grinding thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structures of 3-flavanol, 3,4-flavandiol and(−)-epicatechin.

FIG. 2 depicts an absorbance spectrum from 230 to 550 nm of aproanthocyanidin extract (Fraction 8 of Example 1) after furtherpurification by HPLC with diode array detection using an analytical C-8column eluted with 100% methanol.

FIG. 3A depicts a ¹³C-NMR spectrum of the proanthocyanidin extract ofFraction 8 a of Example 1. FIG. 3B shows the chemical structure of allB-linked proanthocyanidin having 4 or more flavanoid monomer units.FIGS. 3C and 3D depict the ¹³C-NMR spectrum of FIG. 3A in expanded form,where FIG. 3C shows the signal from 60-210 ppm and FIG. 3D shows thesignal from 0-110 ppm. The chemical shifts of the peaks are asindicated.

FIG. 4A depicts a ¹³C-NMR spectrum of the proanthocyanidin extract ofFraction 8 b of Example 1. FIG. 4B shows the chemical structure of anA-linked proanthocyanidin. FIGS. 4C and 4D depict the ¹³C-NMR spectrumof FIG. 4A in expanded form, where FIG. 4C shows the signal from 80-210ppm and FIG. 4D shows the signal from 0-110 ppm. The chemical shifts ofthe peaks are as indicated.

FIG. 5 depicts the electrospray ionization mass spectrum of theproanthocyanidin extract of Fraction 8 b of Example 1.

DETAILED DESCRIPTION OF THE INVENTION

Proanthocyanidins are polyphenolic molecules found in fruits, berriesand other plant material. These molecules belong to the flavanoid familyof compounds. The flavanoid polyphenolics include the catechins,anthocyanins, and proanthocyanidins. Proanthocyanidins are also known asproanthocyanins, leucoanthocyanins, anthocyanogens or procyanidins;these terms can be used interchangeably. For general reviews see,Porter, “Flavans and Proanthocyanidins” in The Flavonoids: Advances inResearch Since 1986 (Harborne, ed.) Chapman and Hall, London, 1993,pp.23-55; Haslam, Chemistry of Vegetable Tannins, Academic Press, NewYork, N.Y., 1966; or Singleton and Esau, Phenolic substances in Grapesand Wine, and Their Significance (1969). Proanthocyanidins are oligomersof 3-flavanols and/or 3,4-flavandiols which dehydrate upon acidtreatment to yield anthocyanins. Proanthocyanidins are also referred toas anthocyanogens in the older literature, i.e. anthocyanin-formers. The3-flavanol monomers of catechin and epicatechin are commonly found inproanthocyanidins. The structures of 3-flavanol, 3,4-flavandiol andepicatechin are provided in FIG. 1.

It is believed that the proanthocyanidins are similar in structure tothe bacterial-binding receptors found on the surface of bladder orkidney cells. These compounds may act, not by killing the bacteriadirectly, but rather by binding bacterial fimbriae and therebypreventing adherence of the bacteria to bladder or kidney cell surfacereceptors. Alternatively, the proanthocyanidins may inhibit biosynthesisof the bacterial fimbriae, without which adherence cannot occur. Nomatter the mechanism, if the bacteria cannot bind to the cells, theycannot multiply, and these are two steps apparently necessary to cause aurinary tract infection. The bacteria are thus carried harmlessly out ofthe body in the urine stream. This anti-adherence property isadvantageous since it eliminates the selective pressure to developantibiotic resistance that can occur during multiplication of bacteriain the presence of antibiotics.

In one embodiment, this invention is directed to a substantiallypurified proanthocyanidin extract from a plant, preferably a plant fromthe family Ericaceae, Rosaceae, Pinaceae or Vitaceae in which theextract is capable of inhibiting agglutination of P-type E. coli but notinhibiting agglutination of type 1 E. coli. More preferably the extractis prepared from a Vaccinium spp. or a Vitis spp. and most preferablyfrom V. macrocarpon in accordance with the methods described herein.Inhibition of agglutination can be measured by known agglutination assaymethods or know methods of measuring bacterial adherence to a surface.These assay methods can also be used to follow purification of theproanthocyanidin extract from plant homogenate material.

For example, A-positive (A+) blood type human red blood cells (HRBC)specifically bind P-type fimbriated E. coli and the extract of theinvention will prevent (i.e., inhibit) agglutination of the A+HRBC inthe presence of P-type bacteria. There are also synthetic P-typereceptor analogs known in the art which can be used to coat solidsubstrates, e.g., latex beads, and then used to assay the extract forits ability to inhibit agglutination of P-type bacteria. Agglutinationassays specific for type 1 fimbriated E. coli are also known and aresimilar to those described above, except that the HRBC can be replacedby guinea pig red blood cells (GPRBC) or by yeast cells, e.g.,Saccharomyces cerevisiae and the agglutination is done in the presenceof type 1-fimbriated bacteria. Details for performing these assays areprovided in the Examples.

Moreover, the extract of the invention is substantially free ofanthocyanins, flavonols, hydrolyzable tannins, alkaloids, lipids,carbohydrates, simple sugars, protein and amino acids, alcohols andorganic acids. The presence or absence of these compounds can bedetermined by standard chemical testing, measures of purity or otherconventional means known in the art. In one method, the extract (orother fraction to be tested) can be spotted on thin-layer chromatography(TLC) plates, developed in an appropriate solvent system and thentreated with a test reagent for a specific class of compounds liketannins, alkaloids, lipids and the like to determine whether thosecompounds were present in the test sample. A variety of useful TLC testsare described in Example 2. Other chemical or purity determining methodsinclude colorimetric detection of anthocyanidin formation, HPLCpurification and ¹H- and ¹³C-NMR spectroscopic identification. Thesemethods are known and some specific examples thereof are also set forthin Example 2.

A proanthocyanidin extract prepared from V. macrocarpon in accordancewith the methods of this invention comprises proanthocyanidin compoundsconsisting of an average of at least four to about seven epicatechinflavanoid units, wherein at least two units are linked together by atleast one A-type interflavanoid linkage and the remaining units areB-type interflavanoid bonds. In a preferred embodiment, theseproanthocyanidin compounds consist of an average of from about four tosix epicatechin flavanoid units.

To simplify reference to specific carbons in the flavanol rings of theproanthocyanidins, carbons at specific positions are referred to by Cn,where the n represents the position number of the carbon in the standardflavanol ring numbering system. Thus the carbon at the eight position ofthe ring is designated as C8, the carbon at the four position of thering is designated as C4, etc. Unless specifically stated otherwise,linkages between ring positions can occur in either the α or β anomericorientation.

An A-type interflavanoid linkage is one which results when the flavanoidunits are joined by two bonds, with one bond occurring between C4 of the“upper or first” unit and C8 of the “lower or second” unit and the otherbond occurring between C2 of the upper unit and the oxygen attached tothe C7 of the lower unit. This linkage leads to the formation of anadditional 6-membered ring. A B-type interflavanoid bond (or linkage) isone which results when the flavanoid units are joined by a single bond.That bond occurs between either the upper unit C4 and the lower unit C8or between the upper unit C4 and the lower unit C6.

In addition to obtaining proanthocyanidin compounds via the extractionprocedure and purification procedures of the invention, the presentinvention also embraces proanthocyanidin compounds prepared by chemicalsynthesis. Methods of synthesizing proanthocyanidins are known in theart, for example, Delcour et at., 1985, J. Chem. Soc. Perkin Trans.I:669-676.

Another aspect of the invention is directed to a method of preparing aproanthocyanidin extract from a plant which comprises (a) homogenizingplant material in an aqueous extraction solvent comprising at leastabout 10% water but no more than about 30% water, about 10% to about 70%acetone, about 5% to about 60% methanol and about 0.05% to about 1%ascorbic acid to prepare a first extract; (b) subjecting that firstextract to further purification steps; and (c) recovering therefrom asubstantially purified proanthocyanidin extract, which is capable ofinhibiting agglutination of P-type E. coli but not type 1 E. coli. Themethod is applicable to plant material from anyproanthocyanidin-containing plant and is preferably used with Vacciniumspp., especially V. macrocarpon or other cranberry species. Thepreferred aqueous extraction solvent comprises about 40% acetone, about40% methanol and about 0.1% ascorbic acid.

Many plants are known to contain proanthocyanidins, and any such plantscan be employed in the method of the invention.Proanthocyanidin-containing plants are members of the Coniferiae classincluding plants from the order Coniferales and particularly from thefamily Pinaceae (including pines); members of the family Filices(including palms); monocot plants form the order Arecales, includingmembers of the families Pandanales, Arales, Najadales, Restionales,Poales (including grains such as sorghum, barley and others), Juncalaes,Cyperales (including cypress), Typhales, Zingiverales, and Liliales(including lilies); dicot plants from the orders Laurales (includinglaurel, cinnamon), Fagales (including oak), Casuarinales, Dilleniales,Malviales (including cotton), Salicales, Ericales (includingcranberries, blueberries, rhododendron), Ebenales, Rosales (includingroses, blackberries and other related berries, apples, peaches, plums),Fabales (including legumes, wysteria), Myrtales, Proteales, Rhamanales(including grapes) and Sapindales. The preferred plants are the dicotsEricaceae, which includes the Vaccinium spp., Rosaceae and Vitaceae,which includes the Vitis spp.; and the conifers of the Pinaceae family.The Vaccinium spp. include, but are not limited to, plants withcranberry-type berries such as V.macrocarpon (cranberry), V. vitis-idaea(mountain cranberry, cow berry, lingonberry) and V. oxycoccus (Europeancranberry); and plants with blueberry fruit such as V. augustifolium(low sweet blueberry), V. ashei (Rabbiteye blueberry), V. corymbosum(high bush blueberry), V. lamarckii (early sweet blueberry) and V.myrtillus (bilberry, European blueberry). The Vitis spp. include, butare not limited to, V. labrusca (Fox grape), V. rotunddifolia(muscadine, scuppenong), V. vinifera (European grape) and allinterspecifc hybrids with other Vitis species.

In one preferred purification scheme, a proanthocyanidin extract of theinvention is prepared by homogenizing plant material from a plant whichcontains proanthocyanidins with an aqueous solution of 40% acetone, 40%methanol and 0.1% ascorbic acid. After homogenization, the extract isclarified by filtration or centrifugation, the pulp is discarded and thesupernatant designated as Fraction 1. The solvents are then removed fromthe supernatant by evaporation and the residue is resuspended indistilled water and subjected to further purification by (1)fractionation on a reverse-phase lipophilic column, such as a C-18column or other comparable column, equilibrated in distilled water andeluted with successive washes of water (yielding Fraction 2), 15%aqueous methanol (yielding Fraction 3), or acidified 100% methanol(yielding Fraction 4 which can be evaporated to dryness before furtherpurification) or (2) by extraction with a non-polar solvent such aspetroleum ether, hexane and the like. The non-polar phase, Fraction 5,is discarded and the aqueous phase, Fraction 6, can also be evaporatedto dryness before further purification.

The next step in the purification process is to resuspend Fraction 4 or6 in 50% aqueous ethanol and apply that solution to a mixedlipophilic-hydrophilic column such as Sephadex™ LH-20 or LH-60(Pharmacia Biotech) equilibrated in 50% ethanol. Other separation mediasuch as MCI gel (Mitsubishi Chemical) or TSK gel (Tosohaus) can besubstituted. The column is washed in the same solvent system until thenon-proanthocyanidin polyphenolic compounds are removed (Fraction 7).Hence complete washing can be monitored by removal of the red colorattributed to the anthocyanin pigment. A purified mixture ofproanthocyanidins is then obtained by eluting the column with 70%aqueous acetone. The resulting mixture (Fraction 8) can be lyophilizedand stored at 4° C. in the dark to minimize oxidation.

As an alternative, Fractions 4 or 6 can be further separated into lowand higher molecular weight proanthocyanidins mixtures by liquid-liquidextraction with ethyl acetate and water prior to further purification.In this case, Fractions 4 or 6 are resuspended in water and extractedfour times with an equal volume of ethyl acetate. The ethyl acetatephases are combined and contain the lower molecular weightproanthocyanidins, whereas the aqueous phase contains the highermolecular weight proanthocyanidins. Both post-extraction phases areevaporated to dryness to remove solvent and then further purified onSephadex LH-20 as described above. The purified material obtained fromfractionating the water-soluble phase on the Sephadex LH-20 column isdesignated as Fraction 8 a. The purifed material obtained fromfractionating the combined ethyl acetate-soluble phases on the SephadexLH-20 column is designated as Fraction 8 b.

As used herein, an aqueous alcohol solution means a solution of waterand alcohol having the specified percentage of alcohol. For example, 15%aqueous methanol means a solution having 15 parts methanol and 85 partswater and 50% aqueous ethanol means a solution having 50 parts ethanoland 50 parts water. Similarly, an aqueous acetone solution means asolution of water and acetone having the specified percentage ofacetone. Further, 100% acidified methanol means methanol containing fromabout 0.005% to about 0.1% acid. The 100% methanol solution of theExamples is methanol and 0.01% ascorbic acid.

In the purification schemes described above, the fractionation stepsemploy column chromatography. However, the skilled artisan willrecognize that the purification steps can also be carried out in batchwith straightforward modifications.

In accordance with the invention, the plant material can be from anypart of the plant and preferably is from a part of the plant rich inproanthocyanidins. For example, plant material includes leaves, fruit(both mature or ripe fruit, and immature or unripe fruit), stems, seeds,bark and roots, and can be used for preparation of the proanthocyanidinextract. In the case of Vaccinium species, the plant material ispreferably from leaves or fruit. For V. macrocarpon, leaves provide therichest source of proanthocyanidins. The mature fruit from V.macrocarpon is red whereas the immature fruit is green.

The solution used to homogenize the plant material is important inobtaining high extraction yields of the proanthocyanidins. The preferredsolution is an aqueous solution of 40% acetone, 40% methanol and 0.1%ascorbic acid. However, the amounts of each component in the extractionsolvent range from 10% to about 70% for acetone, from about 5% to about60% for methanol and from about 0.05% to about 1% ascorbic acid,provided that in all cases there is a minimum of at least about 10%water to a maximum of about 30% water. The ascorbic acid can also rangefrom about 0.05% to about 0.2%.

Acetone is more effective than alcohols alone or aqueous alcohols forsolubilizing and extracting proanthocyanidins from plant tissues(Hagerman, 1988, J. Chem. Ecol. 14 (2): 453-461). If extracting fromleaf tissue, acetone inhibits the interaction between proanthocyanidinsand proteins, preventing binding of the proanthocyanidins to leafproteins during the homogenization process (Hagerman, 1988) andresulting in a higher yield of extracted proanthocyanidins. In addition,because skin waxes, proteins, and polysaccharides are relativelyinsoluble in acetone, these components tend to be left in the pulp.Methanol acts as a mild antioxidant and is important when extractingplant materials which contain large amounts of oxidase enzymes, such asfound in fruit tissue. Water increases the polarity of the solventmixture and enhances the solubility of the sample thereby increasingyields of the proanthocyanidins since these molecules are associatedwith the water-soluble components of the sample. Ascorbic acid acts asan antioxidant by reducing the quinones that are initially formed (Lea,1992, Flavor, Color, and Stability in Fruit Products: The Effect ofPolyphenols. In: Hemingway, R. W. (ed.) Plant Polyphenols. Plenum Press,New York) and also helps to maintain slightly acidic to neutralconditions during the homogenization and extraction process. Alkalineextraction conditions must be avoided since proanthocyanidin moleculesare known to undergo base-catalyzed structural rearrangements at high pHthat cannot be reversed upon exposure to acidic conditions (see, forexample, Laks et al., 1987, J. Chem. Soc., Perkin Trans. I:1875). Upontesting, it was established that such structurally rearranged molecules(which are no longer proanthocyanidins) lose the ability to inhibitadherence of P-type E. coli bacteria to cellular surfaces.

In another embodiment the invention provides methods of preventing ortreating urogenital infections in a mammal by administering acomposition comprising the proanthocyanidin extract or proanthocyanidincompounds of the invention to the mammal in an amount and for a timesufficient to prevent, reduce or eliminate the symptoms associated withsuch infections and thereby lead to amelioration or curing of theinfection. The composition can be a pharmaceutical composition or a foodcomposition and is administered for a time and in an amount sufficientto reduce or eliminate the bacteria associated with urogenitalinfections and thereby ameliorate or cure the infection. Thepharmaceutical composition can also be used to treat diarrhea. Inconnection with preventing infection or disease, to prevent an infectionis not limited to nor necessarily means total prevention of theinfection or disease but also includes uses which lead to action as apreventative for the disease or infection.

As used herein in connection with the various methods of use and inconnection with the pharmaceutical and food compositions of theinvention, the terms “proanthocyanidin compounds” and “proanthocyanidinpolymers” of the proanthocyanidin compositions have the definitions ofone or more of the following compounds or polymers.

There are several type of proanthocyanidin molecules known in the artand any of these are contemplated unless specified otherwise. Theproanthocyanidins include, but are not limited to, procyanidins,prodelphinidins, propelargonidins and profisetinidins.

Hence proanthocyanidin compounds of the invention includeproanthocyanidin compounds capable of inhibiting agglutination of P-typeE. coli but incapable of inhibiting agglutination of type 1 E. coli, andwhich comprises two or more flavanoid monomer units wherein at least twoof said units are linked together by an A-type interflavanoid linkage bybonds between C4 and C8 and between the C2 and the oxygen of C7 of theunits and the remainder of any units are linked to each other by aB-type interflavanoid bond between C4 and C8 or between C4 and C6 of theunits. These molecules can include from two to 10, 20, 30 or even moreflavanoid and preferably have from 4 to 8 monomer units. The moleculeshave at least one A-type interflavanoid linkage, and molecules having afew such linkages, e.g., 2-3 A-type interflavanoid linkages per 10monomer units, as well as molecules having all of the subunits joined byA-type interflavanoid linkages are contemplated. The flavanoid monomerunits can include any of those typically found in proanthocyanidinmolecules and are further defined hereinbelow.

The proanthocyanidin compounds of the invention further includeproanthocyanidin compounds consisting of an average of from at leastfour to about seven epicatechin flavanoid units, and preferably fromfour to six units, wherein at least two of said units are linkedtogether by an A-type interflavanoid linkage by bonds between C4 and C8and between the C2 and the oxygen of C7 of the units and the remainderof the units are linked to each other by a B-type interflavanoid bondbetween C4 and C8 or between C4 and C6 of the units.

The proanthocyanidin polymers of the invention are thoseproanthocyanidin polymers capable of inhibiting agglutination of P-typeE. coli but incapable of inhibiting agglutination of type 1 E. coli. Thepolymers include dimers, trimers, tetramers, pentamers, larger oligomersand long polymers of flavanoid monomer units so long as these polymershave the stated bioactivity.

Hence, the proanthocyanidin compounds and polymers of the invention arecomposed of flavanoid monomers, i.e., polyhydroxyflavan-3-ols, whichinclude but are not limited to, catechin, epicatechin, gallocatechin,epigallocatechin and the like. These monomers can be chemically modifiedat positions not involved in formation of the interflavanoid linkages,including modifications of any hydroxy group not involved inpolymerization, so long as the compounds are capable of inhibitingagglutination of P-type E. coli but incapable of inhibitingagglutination of type 1 E. coli. Such modifications include but are notlimited to substitutions of the following groups at those positions:hydroxy, mercapto, halo, trifluoromethyl, alkyl, alkoxy, alkanoyl,haloalkyl, hydroxyalkyl, alkoxycarbonyl, alkylthio and alkanoyloxy. Asused herein alkyl refers to alkyl chains having from one to six carbonatoms, in any straight or branched configuration. For example, suchmodifications can be found at the C3 and C5 positions, or the C6 or C8position if those atoms are not involved in an interflavanoid linkage.Similarly the aryl ring at the C2 position of the flavanoid monomer canbe aryl or heteroaryl, optionally substituted with any of thesubstituents set forth above. Aryl groups include but are not limited tophenyl, indenyl and naphthyl and the like. Heteroaryl groups include butare not limited to, furyl, imidazolyl, triazolyl, triazinyl, oxazoyl,isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl,tetrazolyl, pyridyl (or its N-oxide), thienyl, pyrimidinyl (or itsN-oxide), indolyly, isoquinolyl (or its N-oxide) and quinolyl (or itsN-oxide). Hence, all such modifications are included herein by referenceto the terms “flavanoid monomer” or “flavanoid monomer unit”.

Further, as used herein in connection with the various methods of usingthe proanthocyanidin extract of the invention and in connection with thepharmaceutical and food compositions described below, the term“proanthocyanidin extract” refers to one or more (a) substantiallypurified plant proanthocyanidin extracts capable of inhibitingagglutination of P-type E. coli but incapable of inhibitingagglutination of type 1 E. coli, including extracts prepared from plantsin the families Ericaceae, Rosaceae, Pinaceae and Vitaceae, andpreferably plants which are a Vaccinium or a Vitis species; and (b) aproanthocyanidin extract prepared by a method of the invention includingextracts prepared from plants in the families Ericaceae, Rosaceae,Pinaceae and Vitaceae, and preferably plants which are a Vaccinium or aVitis species.

Treatment in accordance with the invention renders bacterianon-pathogenic and unable to colonize the urinary tract. Thus, onemeasure of efficacy includes monitoring the reduction or elimination ofurinary bacterial counts associated with such infections during or afterthe course of treatment. Prevention in accordance with the inventiondoes not mean complete prevention of infection in any particularindividual but rather means a statistical reduction in the incidence ofurogenital infections in a population sample.

Pharmaceutical compositions comprise proanthocyanidin extracts orproanthocyanidin compounds together with a pharmaceutically acceptablecarrier. The pharmaceutical composition can be provided, e.g., in tabletor liquid form, oral rinse, douche, topical formulation, toothpaste oras an additive for a beverage or other food item, especially as anadditive for a cranberry juice beverage (or cranberry-juice containingbeverage) to allow enhancement of the cranberry juice's known benefitsfor preventing and treating urinary tract infections.

Specific pharmaceutical compositions of the invention comprise aproanthocyanidin extract and a pharmaceutically-acceptable carrier,wherein the extract is capable of inhibiting agglutination of P-type E.coli and not capable of inhibiting agglutination of type 1 E. coli aswell as any of the extracts prepared in accordance with a method of theinvention. Another specific pharmaceutical composition of the inventioncomprises one or more proanthocyanidin compounds and apharmaceutically-acceptable carrier, wherein the compounds areproanthocyanidin compounds consisting of an average of from at leastfour to about twelve epicatechin flavanoid units, wherein each unit islinked to the next by B-type interflavanoid bonds or proanthocyanidinoligomers consisting of an average of from at least four to about sevenepicatechin flavanoid units, wherein at least two units are linkedtogether by at least one A-type interflavanoid linkage and the remainingunits are B-type interflavanoid bonds.

The proanthocyanidin compositions of the invention can be used directlyas food additives or mixed with a consumable carrier to be used as afood additive or food composition. One food additive of the inventioncomprises a proanthocyanidin extract of a Vaccinium species and aconsumable carrier, wherein the extract is capable of inhibitingagglutination of P-type E. coli and not capable of inhibitingagglutination of type 1 E. coli. Another food additive of the inventioncomprises one or more proanthocyanidin compounds and a consumablecarrier, wherein the compounds are proanthocyanidin oligomers consistingof an average of from at least four to about seven epicatechin flavanoidunits, wherein at least two units are linked together by at least oneA-type interflavanoid linkage and the remaining units are B-typeinterflavanoid bonds.

The food compositions of the invention thus contain one of theproanthocyanidin compositions of the invention in admixture withlivestock feed, domestic animal feed or with a consumable food product.Those food compositions which contain livestock feed are for cattle,pigs, turkeys, chickens and the like. Those food compositions whichcontain domestic animal feed are for dogs, cats, horses and the like.Those food compositions which contain a consumable food product are formammals, preferably for humans and primates. The food compositions,especially beverages, can be used as therapeutics to prevent or treaturogenital infections. Alternatively, the food compositions can begeneral consumables, for example, ground meat or other meat product,beverages, especially juice beverages, whether or not pasteurized, grainproducts, fruit products and the like. Beverages which contain cranberryjuice are preferred.

As used herein a consumable food product includes, but is not limitedto, a cranberry-containing food product, a beverage, ground meat or anyother edible product to which the proanthocyanidins can be added.Cranberry-containing food product include dried cranberries, sweetenedand dried cranberries, flavored fruit pieces, cranberry sauces,cranberry jellies, cranberry relishes, cranberry juices or any otherbeverage or product containing cranberry juice and wine made from orwith cranberries. Beverages include unpasteurized juice or pasteurizedjuice.

The preferred dosage range of proanthocyanidin composition is from about1 mg to about 500 mg per day, preferably from about 10 to about 250 mgper day and more preferably from about 25 to 100 mg per day of extract,compound or polymer. Such dosages can be present in tablets or otherpharmaceutical compositions as well as in the food compositions of theinvention including beverages and other food items. If the beverage iscranberry juice, the dosage can be adjusted, if desired, to account forthe proanthocyanidins already present in the juice. This adjustment canreadily be made by one of skill in the art by determining the amount ofproanthocyanidins present in the juice and making the appropriatecompensation or desired supplementation. Similarly, if the patient isbeing treated with both cranberry juice and a proanthocyanidinsupplement, then the dosage of proanthocyanidins needed to achieve ananti-adherence effect (or other effect such as a lessening of symptoms)could be reduced.

Use of a Vaccinium-derived (or other plant-derived) extract orpharmaceutical composition can be beneficial in places where cranberryjuice is not available (third-world countries, etc.). Moreover, somepeople, especially the elderly, that suffer recurrent urinary tractinfections cannot tolerate cranberry juice due to its high sugar andacid content and a proanthocyanidin-containing pharmaceuticalcomposition would be an effective and desirable alternative.

Another aspect of the invention relates to methods of reducing P-type E.coli contamination in ground meat, preferably from cattle but also frompigs, chickens, turkeys or other livestock sources of ground meat. Forexample, raw meat can be obtained from a livestock animal and a foodadditive of the invention can be mixed with the raw meat before orduring preparation of ground meat from the raw meat. Another way toreduce P-type E. coli contamination in ground meat comprises feeding afood composition of the invention to a livestock animal and preparingground meat from that animal. The ground meat can be prepared solelyfrom the meat of animals which have been fed aproanthocyanidin-containing food composition or can also contain meatfrom animals who did not feed on any proanthcyanidin-containing foodcompositions. In the latter case, the ground meat can be prepared, forexample, using a proportion of raw meat from proanthocyanidin-fedanimals sufficient to reduce the agglutination of P-type E. colimicroorganisms in said ground meat relative to ground meat prepared onlyfrom raw meat of a similar livestock animal who has not been fed aproanthocyanidin-containing food composition of the invention.

The livestock animals may not need to be fed aproanthocyanidin-containing food composition during the entire durationof their lifetime. It may be beneficial and cost effective to feed theanimals the food composition of the invention for a period prior toslaughter, e.g., for a few days or weeks or for as long as severalmonths depending on the animal and age at which the animal is destinedfor slaughter.

In another embodiment, the present invention provides a method ofinhibiting adherence of P-type E. coli to a surface which comprisescontacting said bacteria with at least one proanthocyanidin composition,prior to or concurrently with contacting said bacteria with saidsurface. The surface can be any substance or material, synthetic orbiological, where it is desired to prevent bacterial contamination,accumulation or infection. The surface can also be or constitute abiofilm. In a preferred embodiment the surface is a cellular surfacesuch as an uroepithelial cell surface, cells exposed in a wound or onthe skin or another surface such as teeth or a prosthetic device orimplant or a biofilm on any of these objects. The proanthocyanidincomposition used in this method can be any proanthocyanidin extractcompound or polymer of the invention or a pharmaceutical productcontaining any of the foregoing.

The proanthocyanidin compositions of the invention can also be used forreducing or treating infection after surgery, treating topical wounds oracne, or preventing or eliminating oral infection by administering apharmaceutical composition of the invention to a site of infection orpotential infection in a patient. The pharmaceutical composition isadministered to the patient in accordance with the treatment beingrendered. For example, it can be applied to a surgical incision or otheropening as a liquid, topical cream or by any other suitable deliverymeans. For topical wounds, the pharmaceutical composition can be atropical cream, salve or spray. Oral infection can be treated bybrushing with a toothpaste or by using a oral rinse or mouth washformulated with proanthocyanidins in accordance with the invention.

Yet another aspect of the invention is directed to a method of detectingP-type reactive bacteria in a body fluid sample which comprises (a)contacting a body fluid sample with a P-type receptor specific assayreagent for a time and in an amount to allow binding of any P-typereactive bacteria which may be present in the sample to the reagent,wherein the reagent comprises a solid-phase substrate coated with one ormore proanthocyanidin compositions of the invention; and (b) determiningwhether P-type reactive bacteria are present in said sample by assessingthe degree of agglutination in said sample. The presence of P-typereactive bacteria, especially P-type E. coli, are present ifagglutination occurs in the sample. Various modifications of the assayare contemplated including sandwich type assays and inhibition ofagglutination assays.

Another embodiment of the invention provides a kit for use in detectingP-type reactive bacteria in a body fluid sample. The compartmentalizedkit comprises a container adapted to contain a P-type receptor specificassay reagent which comprises a solid-phase substrate coated with one ormore proanthocyanidin compositions of the invention. The kit can alsocontain diluent for the sample or the reagent. Further, the kit may beadapted, if desired, for conducting the assay directly in the containercontaining the reagent. Alternatively, the kit can contain a containeradapted for conducting the assay, and especially for multiple serialdilutions of the sample or reagent as appropriate.

It is to be understood and expected that variations in the principles ofinvention herein disclosed in an exemplary embodiment may be made by oneskilled in the art and it is intended that such modifications, changes,and substitutions are to be included within the scope of the presentinvention.

EXAMPLE 1 Preparation of Proanthocyanidin Extract

Leaves or fruit (red—mature and green—immature) (10 g fresh weight) ofthe plant Vaccinium macrocarpon (cranberry) were washed, dried, andhomogenized with 60 mL of extraction solvent (40% acetone, 40% methanol,20% water, 0.1% ascorbic acid) in a blender for 10 min. The homogenizedslurry was forced through 8 layers of cheesecloth, the pulp discarded,and the crude extract filtered or centrifuged at 5,000 rpm for 15minutes to remove insoluble particulate matter. The clarifiedsupernatant, designated as Fraction 1, was evaporated to dryness underreduced pressure to remove solvents and was resuspended in 17 mLdistilled water. Lipids, waxes, sugars, and acids were removed fromFraction 1 by application of the fraction to a reverse-phase lipophiliccolumn (C-18) that had been preconditioned with 1 column volume ofmethanol followed by 1 column volume of water. The column wassuccessively washed with the indicated solvents as follows: Sugars wereremoved by washing with 2 column volumes of distilled water to produceFraction 2. Acids were removed by washing with 2 column volumes of 15%aqueous methanol to produce Fraction 3. The polyphenolic compounds werecompletely eluted with 3 column volumes of 100% methanol acidified with1% acetic acid to produce Fraction 4. Fraction 4 was evaporated todryness before further purification.

As an alternative, the lipids and skin waxes present in Fraction 1 wereremoved by extracting Fraction 1 three times with an equal volume ofpetroleum ether. The petroleum ether-soluble phase, designated asFraction 5, was discarded, and the aqueous phase, designated as Fraction6, containing sugars, acids, and polyphenolic material was evaporated todryness to remove all traces of solvent; the remaining sugars and acidswere removed in subsequent steps.

For those samples analyzed by NMR (see Example 2, section D), Fraction 6was subjected to an additional extraction step prior to furtherpurification. To carry out this extraction, the aqueous phase designatedas Fraction 6 was not evaporated to dryness but rather its volume wasreduced and the resultant solution was extracted four times with anequal volume of ethyl acetate. The water-soluble fraction was reduced involume and subjected to further purification on an Sephadex LH-20 columnas described below. This purified preparation is designated as 8 a andcontains the water-soluble proanthocyanidins. The ethyl-acetate solublefractions were combined, evaporated to dryness and further purified on aSephadex LH-20 column as described below. This purified preparation isdesignated as 8 b and contains ethyl acetate-soluble proanthocyanidins.

To separate the proanthocyanidins in Fraction 4 or 6 from otherpolyphenolic compounds, such as anthocyanins and flavonols, the driedfraction was resuspended in a minimum volume of 50% aqueous ethanol andapplied to a glass column containing hydroxypropylated cross-linkeddextran beads (Sephadex LH-20, Pharmacia Biotech) that had beenequilibrated overnight in 50% aqueous ethanol. The column was washedwith up to 10 column volumes of 50% aqueous ethanol or until all redcolor had been removed. This wash eluate, designated as Fraction 7, wascomposed of non-proanthocyanidin polyphenolic compounds (flavonols,anthocyanin pigments). When Fraction 6 was applied to the LH-20 column,the eluate also included any sugars and acids remaining from solventpartitioning. The proanthocyanidins, designated as Fraction 8, were theneluted from the column with up to 8 column volumes of 70% aqueousacetone, freeze-dried and stored in the dark at 4° C. to minimizeoxidation. The freeze-dried material was weighed and the mg ofproanthocyanidins (dry weight) per g fresh weight of unfractionatedextract was calculated to determine the relative concentration ofproanthocyanidins extracted from cranberry leaves and fruit (ripe andunripe) (Table 1).

TABLE 1 Proanthocyanidin Concentration in Cranberry Organs Concentrationof Proanthocyanidin Source Extract (mg/g)^(a) Leaves 21.16 Fruit(unripe) 12.14 Fruit (ripe) 10.72 ^(a)Extract from Fraction 8; mg dryweight proanthocyanidin/g fresh weight source.

EXAMPLE 2 Composition of Proanthocyanidin Extract

A. Thin Layer Chromatography

Chemical testing was undertaken to demonstrate that Fraction 8 containedpurified proanthocyanidins and was free from other constituents. Thinlayer chromatography (TLC) was performed on precoated silica gel plateswith a fluorescence indicator (Fisher Scientific). Fraction 8 from ripefruit was spotted (+μl) onto TLC plates and developed with benzene-ethylformate-formic acid (2:7:1) to determine whether a particular class ofcompounds was present. Each TLC plate was sprayed and/or treated withthe chemical reagents for the indicated compounds as described below.After treatment, the TLC plates were examined and noted for developmentof spots and the color of those spots. The results are shown in Table 2and demonstrate that the extracts are substantially pureproanthocyanidins.

The chemical reagent tests for specific compounds and expected resultswere as follows:

Tannins (general): A solution of 1% ferric chloride in 0.5 N HCl wassprayed on the TLC plate. Tannins yield a green-brown spot.

Procyanidins: A solution of 1% ethanolic vanillin and a solution ofmethanol:HCl (8:2, v/v) were successively sprayed on the TLC plate. Abright red spot indicates presence of procyanidins.

Hydrolyzable tannins: The TLC plate was sprayed with a saturated aqueoussolution of potassium iodate. The galloyl esters produce a red to pinkspot whereas gallic acid produces an orange-red spot.

Alkaloids: A mixture of 5% I₂ in 10% KI solution was made and then 2parts of solution were mixed with 3 parts water and 5 parts 2 N aceticacid before spraying the TLC plate. Appearance of a spot(s) indicatesthat alkaloids are present.

Carbohydrates: A solution of 3% anthrone in boiling glacial acetic acidwas sprayed on the TLC plate followed by a spray of ethanol:phosphoricacid:water (20:3:1, v/v). The plate was heated 5 min. at 110° F. Ketosesand oligosaccharides produce a yellow spot.

Simple sugars: The spray consisted of 1.23 g p-anisidine and 1.66 gphthalic acid dissolved in 100 mL methanol. Hexose sugars yield a greenspot whereas pentose sugars yield a red-violet spot.

Protein/amino acids: A solution of 2% vanillin in n-propanol was sprayedon the TLC plate. The plate was heated for 10 min and observed under UVillumination at 254 nm. A second spray of 1% KOH in ethanol, followed byheating the plate again produced various colored spots.

Lipids: The spray consisted of 0.1 g ferric chloride and 7 gsulfosalicylic acid dissolved in 25 mL water and then diluted to 100 mLwith 95% ethanol. With fluorescent illumination lipids appear as whitespots visible on purple background.

Organic acids: Solution A was prepared by dissolving 0.17 g silvernitrate in 1 mL water, adding 5 mL ammonia and diluting to 200 mL withethanol. Solution B consisted of 6.5 g pyrogallol in 100 mL ethanol. TheTLC plates were sprayed with solution A followed by solution B.Appearance of a spot(s) indicates the presence of organic acids.

Alcohols: The spray was prepared by dissolving 3 g vanillin in 100 mLabsolute ethanol and adding 0.5 mL concentrated sulfuric acid. Theplates were sprayed and heated at 120° F. Higher alcohols and ketonesproduce a blue-green spot.

TABLE 2 Composition of Fraction 8 Determined by TLC with SelectiveChemical Reagents Compound TLC Plate Detected 1 2 3 4 5 6 7 8 9 10Tannin + + + + + + + + + + Procyanidin + + + + + + + + + + Hydrolyzable− − − − − − − − − − tannin Alkaloid − − − − − − − − − − Carbohydrate − −− − − − − − − − Simple sugar − − − − − − − − − − Protein − − − − − − − −− − Lipid − − − − − − − − − − Alcohol − − − − − − − − − − Organic acid −− − − − − − − − −

B. Anthocyanidin Formation

Two additional chemical tests were used to confirm the proanthocyanidincomposition of Fraction 8: (1) the formation of anthocyanidins aftertreatment with n-butanol-HCl (Govindarajan & Mathew, 1965,Phytochemistry 4:985-988), and (2), color formation with absorbance at500 nm following sulfuric acid-catalyzed condensation of vanillin withthe phloroglucinol ring (Swain & Hillis, 1959, J. Sci. Food Agric.10:63-68).

C. HPLC

High performance liquid chromatography (HPLC) was also employed todemonstrate the purity of the proanthocyanidin extract, Fraction 8 frommature red fruit. The extract was injected onto an analytical C-8 column(Zorbax 300SB 4.6 mm×25 cm, MacMod Analytical) and run using a gradientpump with a mobile phase of 100% methanol. Absorbance was measured usinga photodiode array detector (Dionex Corp.) that detected all wavelengthssimultaneously.

The absorbance spectrum (230 to 550nm) of Fraction 8 showed absorptionat 280 nm, which is characteristic of proanthocyanidins (FIG. 2). Therewas no absorption in the 360 or 550 nm range, which would be indicativeof the presence of flavonols or anthocyanin compounds, respectively.

D. ¹³C-NMR Spectroscopy

The ¹³C-NMR spectrum of Fraction 8 a and 8 b (Example 1) was performedon a Bruker Avance 300 model at 75 MHZ and referenced to TMS. Spectrawere obtained using a 5 mm quard nuclear probe with a zgpg 30 pulsesequence with proton wartz decoupling and acquisition time set at 0.865seconds with a delay time of 0.50 seconds. The total number of scans was7500.

Fraction 8 a (the water-soluble fraction) consisted of aproanthocyanidin mixture where all signals of any significance could beattributed directly to the constitutive procyanidin flavanoid units(FIG. 3). Procyanidin flavanoid units are characterized by a three ringsystem consisting of an aromatic A-ring based on phloroglucinolhydroxylation pattern, a pyran C-ring fused to the A-ring and anappending catechol B-ring attached to C2 of the pyran ring. The absenceof other carbon resonances in the spectrum indicated that the sampleconsisted of pure procyanidin derivatives. The ¹³C-NMR spectrum ofFraction 8 a was fully in accord with this chemical structure.

The oxygenated aromatic carbons C5, C7, and C8 a of the A-ring wereevident in the low field region of the spectrum (150-158 ppm), whilethose similarly substituted carbons (C3′ and C4′) of the B-ring wereobserved at 142-145 ppm, and those of the aliphatic pyran ring (C3) werefound higher upfield at 64-74 ppm. The observation of the remainingcarbon resonances at 94-108 ppm for C4 a, C6 and C8 of the A-ring, at114-132 ppm for C1′, C2′, C5′, and C6′ of the B-ring, and at 27-38 ppmfor C4 of the C-ring fully accounted for all the carbon atoms of theflavanoid constitutive units. These flavanoid units were linked togetheras proanthocyanidins as confirmed by the presence of diagnostic carbonpeaks at about 36 ppm and 106 ppm for the inter-flavanoid-linked carbonsC4 and C6/C8, respectively. The ratio of the 66 ppm peak area (C3 ofbottom unit) to that of the 72 ppm peak area (C3 of extender units) isabout 1:5.4 suggesting that the average chain length is between 6 and 7epicatechin units.

Procyanidins consist of catechin and/or epicatechin flavanoid units. Thedistinguishing feature between a catechin and epicatechin unit is thelow field position (80-84 ppm) of the C2 carbon chemical shiftassociated with the 2,3-trans configuration of the catechin unitcompared to the more upfield position (75-79 ppm) of the C2 carbonchemical shift associated with the 2,3-cis configuration of theepicatechin unit. Fraction 8 a exhibits only the higher field signals(75-79 ppm), indicating that these procyanidins are predominantlyepicatechin units.

The ¹³C-NMR spectrum of Fraction 8 b (FIG. 4), representing the purifiedethyl acetate-soluble fraction, was fully consistent with that of aproanthocyanidin oligomer where all the major carbon signals could bedirectly attributed to the constitutive flavanoid units as detailedabove for the proanthocyanidins from the water-soluble fraction.However, there were some significant differences in the fine structureof the carbon chemical shifts between the two ¹³C-NMR spectra, the mostapparent being the appearance of new multiple peaks between 78 ppm and82.5 ppm consistent with the C2 of terminating flavanoid units and theupfield position of some hydroxylated A-ring carbons at 150 to 152 ppm.The latter shifts are characteristic of the oxygenated carbons of theA-ring involved in the A-type linkages. Another probable indication ofthe presence of the A-type linkages in the sample was the unexpectedsignal size at 66-67 ppm usually attributed to the C3 of the terminatingflavanoid unit, so much so that this signal area was comparable to thatof the extender C3 at 72.8 ppm. This observation would have suggested adimeric structure but the broad nature of the signals ruled this outbecause in A-type linkages, the new ether bond formed at C2 causes anupfield shift of the C3 signal of the extender unit to the generalregion around 67 ppm. This shift can account for the additional size ofthe signal observed in that region.

Additional evidence for the presence of the A-type linkage was obtainedfrom H,C-COSY NMR data (HMQC). In A-type linkages the chemical shift ofC4 of the “upper” C-ring coincides with that of the C4 from the terminalunit in the proanthocyanidin polymer, however, these two carbons can bedistinguished by the chemical shift of their respective attached protonsin which the chemical shift of the proton on the A-type C4 is generallyobserved downfield between 4 to 5 ppm while the proton of the C4extender unit is observed upfield around 3 ppm. The HMQC spectrum ofFraction 8 b showed that those carbons with a chemical shift around 29.4ppm in the ¹³C-NMR spectrum had proton resonances at 4.23 ppm and 2.90ppm and thus indicated the presence of an A-type linkage in thisproanthocyanidin oligomer.

Moreover, the peak area of the C3 chemical shifts around 67 and 72 ppmwere in the ratio of about 1 to 1.29, suggesting that there is oneterminal unit to just over 3 extender units with the chemical shift fromone of the extender units being located in the C3 terminal unit becauseof the presence of an A-type linkage.

This structure was further confirmed by electrospray ionization massspectrometry (ESI-MS) as shown in FIG. 5. Two main moleculer negativeion peaks were observed at m/z 1151 and 1439 and are attributable to atetramer and pentamer, respectively, containing one A-typeinterflavanoid linkage. In addition, the smaller peak at higher mass m/z1725 suggested the sample also contained a small amount of aproanthocyanidin hexamer with two A-type interflavanoid linkages.Negative ion peaks observed at lower mass were also fully consistentwith these results where the peak at m/z 862.8 is a trimer with oneA-type linkage (charge −1) or a hexamer with one A-type linkage (charge−2), the peak at m/z 719.4 is a pentamer (charge −1) with one A-typelinkage and the peak at m/z 575.3 is a tetramer (charge −1) with oneA-type linkage.

EXAMPLE 3 Bioactivity of Proanthocyanidin Extracts

A. Bacterial Strains

Sixty E. coli strains were isolated from the urine of human and animal(dog and cat) patients suffering from urinary tract infections. Afterdetermining the fimbrial type of each strain, 5 strains specific forP-type fimbriae, and 5 strains specific for type 1 fimbriae wereselected for use in bioactivity testing. Bacterial strains weresubcultured in tryptose broth at 37° C. for 16 h. P-type strains weretransferred to colonization factor agar (CFA) plates and grow novernight at 37° C. to enhance production of P-type fimbriae. Strainswere harvested by centrifugation, washed once and suspended inphosphate-buffered saline solution (PBS) at pH 7.0 at a concentration of5×10⁸ bacteria/mL. The individual phenotypic strains were tested in eachof the following bioassays and did not differ significantly in theirability to agglutinate red blood cells or yeast. Therefore, mixtures ofP-type strains were used to test for P-type bioactivity, and mixtures oftype 1 strains were used to test for type 1 bioactivity. Strains werekept frozen at −70° C. in tryptose broth (30% glycerol) for long-termstorage.

B. Inhibition of Adherence Bioassay for P-type E. coli

1. Hemagglutination Assay

Red blood cells donated by human volunteers with A+ blood type (HRBC)were suspended at a concentration of 3% HRBC. Fractions to be tested forbioactivity were suspended in PBS and adjusted to neutral pH with 1 MNaOH. The bioassay should be performed under neutral conditions,otherwise the assay leads to false-positive readings at low pH (<4) andhigh pH (>8) in the bioassay (Table 3).

Dried Fractions 1-8 from Example 1 were rehydrated in PBS, neutralized,and tested for ability to inhibit agglutination of HRBC. Dilutedfractions (30 μl) were incubated with 10 μl of a P-type bacterialsuspension in a 24-well polystyrene plate for 10 min on a rotary shaker.HRBC (10 μl) were added to each well and incubation continued for 20 minon a rotary shaker. Controls which showed no agglutination or completeinhibition of agglutination included (1) bacteria only (2) HRBC only,(3) bacteria and test fraction and (4),bacteria, HRBC and epicatechintrimer, a proanthocyanidin standard. The control to show agglutinationconsisted of bacteria and HRBC.

The ability of each fraction to inhibit bacterial adherence in thisassay was assessed microscopically and is provided in Table 4 under thecolumn “HRBC”. A “+” indicates that the fraction inhibited P-typeadherence and a “−” indicates that no adherence was observed.

To compare the bioactivity of proanthocyanidin extracts from leaves andfruit (mature and immature), serial (2-fold) dilutions of Fractions 1and 8 were made using equivalent amounts of material obtained from 1 gof starting material (fresh weight). Thus, for ripe fruit 10.72 mg ofFraction 1 or Fraction 8 were dissolved in 1 mL PBS to prepare theundiluted solution subjected to the serial dilution. Similarpreparations were made for unripe fruit using 12.14 mg and for leavesusing 21.16 mg. The relative agglutination in the dilution series wasrecorded by microscopic observation of the agglutination reaction andscored on a scale of 0 to 3 with a score of 0 representing 95-100% ofHRBC agglutinated (no inhibition of agglutination by the test fraction),a score of 1 representing 50-95% of HRBC agglutinated, a score of 2representing 5-50% of HRBC agglutinated, and a score of 3 representingless than 5% of the HRBC agglutinated, i.e., total inhibition ofagglutination by the test fraction. The results provided in Table 5demonstrate that the cranberry plant leaves contain moreproanthocyanidins than fruit on a fresh weight basis and are more activein inhibiting P-type E. coli to HRBC than the proanthocyanidinsextracted from the cranberry fruit.

2. Latex Bead Agglutination Test

The fractions were also tested in the P-type receptor bioassay of de Manet al., 1987, J. Clin. Microbiol. 25(2):401-406. Dried Fractions 1-8were dissolved in PBS, neutralized, and mixed with P-type bacterialsuspensions (30 μl fraction to 10 μl of bacterial suspension) on apolystyrene 24-well plate and incubated for 10 min on a rotary shaker.Latex beads coated with a synthetic P-type receptor analog (Galα1→4Galβ)(BACH test, Kabi Vitrum, Stockholm, Sweden) suspended in water at aconcentration of 5×109 beads/mL were added (10 μl) to each well andincubation continued for 20 min on the rotary shaker. Controls whichshowed no agglutination or complete inhibition of agglutination included(1) bacteria only (2) bacteria and uncoated beads, (3) bacteria and testfraction, (4) uncoated beads and test fractions, and (5) bacteria,coated beads and epicatechin trimer. The control to show agglutinationconsisted of bacteria and coated beads. The relative ability of eachfraction to inhibit bacterial adherence in this assay was assessedmicroscopically and is provided in Table 4 under the column“P-receptor”. A “+” indicates that the fraction inhibited P-typeadherence and a “−” indicates that no adherence was observed.

C. Inhibition of Adherence Bioassay for Type 1 E. coli

1. Hemagglutination Assay

The inhibition of adherence of type 1 E. coli by the various fractionswas tested according to hemagglutination assay for P-type bacteria,except guinea pig red blood cells (GPRBC) were used in place of HRBC.The results are shown in Table 4 under the column GPRBC using the samescoring system as described above.

2. Yeast Agglutination Assay

The fractions were also tested in the bioassay of Eschdat et al., 1978,Biochem. Biophys. Res. Commun. 85:1551-1559, using yeast cells. Yeastcultures of Saccharomyces cerevisiae were grown in Sabouraud dextrosebroth and incubated for 24 hr at 38° C. Yeast cells were transferred toSabouraud dextrose agar plates and grown under the same conditions.Cells were harvested, washed and suspended in PBS at a concentration of4×108 cells/mL. Ten μl of type 1-fimbriated E. coli were mixed withneutralized test fractions (30 μl) in a 24-well polystyrene plate andincubated for 10 min on a rotary shaker. Yeast cell suspension (10 μl)was added to each well and incubated for 5 min. Controls which showed noagglutination or complete inhibition of agglutination included (1)bacteria only (2) yeast only, (3) bacteria and test fraction and (4)bacteria, yeast and 1% D-mannose. The control to show agglutinationconsisted of bacteria and yeast. The results are shown in Table 4 underthe column “Yeast Cells” using the same scoring system as describedabove.

TABLE 3 Agglutination Response as a Function of pH for P-type E. coliBuffer pH Agglutination Response 2 − 3 − 4 +/− 5 + 6 + 7 + 8 +/− 9 − 10−

TABLE 4 Inhibition of Agglutination of P-type and Type 1 E. coli byExtracted Cranberry (V. macrocarpon) Fractions HRBC P-Receptor GPRBCYeast Cells 1- Clarified Whole + + + + Extract 2- Sugars − − + + 3-Acids − − − − 4- Total Polyphenolics + + − − 5- Lipids, Waxes − − − − 6-Polyphenolics, Sugars, + + + + Acids 7- Flavonols, − − − − Anthocyanins8- Proanthocyanidins + + − − Controls 1% D-Mannose − − + + EpicatechinTrimer + + − −

TABLE 5 Relative Inhibition of P-type E. coli-induced HRBC Agglutinationby Cranberry Extracts Unfractionated Extract Proanthocyanidin ExtractRipe Unripe Ripe Unripe Dilution Fruit Fruit Leaves Fruit Fruit Leaves1:1 3 3 3 3 3 3 1:2 3 3 3 3 3 3 1:4 3 3 3 3 3 3 1:8 1 2 3 3 3 3 1:16 0 03 3 3 3 1:32 0 0 2 2 3 3 1:64 0 0 0 2 3 3 1:128 0 0 0 1 2 3 1:256 0 0 00 0 3 1:512 0 0 0 0 0 0

We claim:
 1. A proanthocyanidin composition comprising proanthocyanidincompounds having an average of from at least four to about sevenepicatechin flavanoid units, wherein each proanthocyanidin compound hasat least two of said units are linked together by an A-typeinterflavanoid linkage by bonds between C4 and C8 and between C2 and theoxygen of C7 of the units, and the remainder of the units are linked toeach other by a B-type interflavanoid bond between C4 and C8 or betweenC4 and C6 of the units.
 2. A food composition comprising a consumablecarrier in admixture with the proanthocyanidin composition of claim 1.3. The composition of claim 1, wherein said compounds consist of anaverage of four, five or six epicatechin flavanoid units.
 4. Apharmaceutical composition comprising the proanthocyanidin compositionof claim 1 or 3 and a pharmaceutically acceptable carrier.
 5. The foodcomposition of claim 2 wherein said consumable carrier is livestockfeed.
 6. The food composition of claim 2 wherein said consumable carrieris domestic animal feed.
 7. The food composition of claim 2 wherein saidconsumable carrier is a consumable food product.
 8. The composition ofclaim 7, wherein said consumable food product is a cranberry-containingfood product.
 9. The composition of claim 8, wherein saidcranberry-containing food product is a dried cranberry, a sweetened anddried cranberry, a flavored fruit piece, a sauce, a jelly, a relish,juice, wine or a cranberry juice-containing product.
 10. The compositionof claim 7, wherein said consumable food product is a beverage.
 11. Thecomposition of claim 10, wherein said beverage comprises cranberryjuice, unpasteurized juice or pasteurized juice.
 12. The foodcomposition of claim 7, wherein said consumable food product is groundmeat.